基于多重PCR方法的新疆褐牛乳中乳房炎主要致病菌的检测

Detection of main pathogenic bacteria of mastitis in milk of Xinjiang Brown cattle based on multiplex PCR

为了能同时检测到新疆褐牛乳房炎乳中无乳链球菌、金黄色葡萄球菌、沙门氏菌和大肠杆菌四种致病菌,试验根据GenBank中的金黄色葡萄球菌nuc、大肠杆菌phoA、无乳链球菌ef-tu和沙门氏菌invA基因保守序列设计了4对特异性引物,提取新疆褐牛77份乳样DNA,采用单一PCR和多重PCR方法进行扩增,统计琼脂糖凝胶电泳结果并计算相应感染率,比较两种检测方法的符合率,并检测多重PCR方法在牛场中的应用效果。结果表明:单一PCR方法中以大肠杆菌和金黄色葡萄球菌感染为主,感染率分别为93.5%和76.6%。多重PCR方法中,以大肠杆菌+金黄色葡萄球菌+无乳链球菌的混合感染为主,感染率为24.7%;其次是大肠杆菌+金黄色葡萄球菌的混合感染,感染率为16.9%。对两种检测方法的结果进行比较,大肠杆菌+金黄色葡萄球菌+无乳链球菌及大肠杆菌+金黄色葡萄球菌混合感染的符合率分别为95%和93%;大肠杆菌+沙门氏菌+无乳链球菌的混合感染符合率为0,但多重PCR检出数量仅有2份;其余致病菌混合感染的符合率为100%。结合牧场的DHI报告及兽医诊断确认新疆褐牛中的乳房炎主要致病菌为大肠杆菌的单一感染和无乳链球菌+大肠杆菌的混合感染,感染率分别为53.8%、30.8%。说明该多重PCR方法可用于新疆褐牛乳房炎的诊断。

In order to simultaneously detect the infection of four pathogenic bacteria Streptococcus agalactiae,Staphylococcus aureus,Salmonella and Escherichia coli in mastitis in Xinjiang Brown cattle milk,in this experiment four pairs of specific primers were designed according to the conserved sequences of the genes of Staphylococcus aureus nuc,Escherichia coli phoA,Streptococcus agalactiae ef-tu and Salmonella invA in GenBank.DNA from 77 milk samples of Xinjiang Brown cattle was extracted;single PCR and multiplex PCR were used for the amplification;the results of agarose gel electrophoresis were counted and the corresponding infection rate was calculated.The coincidence rate of the two detection methods was compared,and the application effect of multiplex PCR method in cattle farm was tested.The results showed that Escherichia coli and Staphylococcus aureus were the main infections in the single PCR method,and the infection rates were 93.5%and 76.6%,respectively.In the multiplex PCR method,the mixed infection of Escherichia coli+Staphylococcus aureus+Streptococcus agalactiae was the main infection and the infection rate was 24.7%,followed by the mixed infection of Escherichia coli+Staphylococcus aureus with the infection rate of 16.9%.Comparing the results of the two detection methods,the coincidence rates of Escherichia coli+Staphylococcus aureus+Streptococcus agalactiae and Escherichia coli+Staphylococcus aureus mixed infection were 95%and 93%.The coincidence rate of mixed infection of Escherichia coli+Salmonella+Streptococcus agalactiae was 0,but only 2 samples were detected by multiplex PCR.The coincidence rate of mixed infection of other pathogenic bacteria was 100%.Combined with the DHI report of the pasture and the veterinary diagnosis,the main pathogenic bacteria in Xinjiang Brown cattle milk samples confirmed as clinical mastitis were single infection of Escherichia coli and mixed infection of Streptococcus agalactiae+Escherichia coli;the infection rates were 53.8%and 30.8%respectively.The results suggested that the multiplex PCR method could be used for the diagnosis of Xinjiang Brown cattle mastitis.